Erratum in

Dev Cell. 2012 Nov 13;23(5):1081.

Abstract

To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive chromosome movements. How kinetochores associate with depolymerizing microtubules, which undergo dramatic structural changes forming curved protofilaments, has yet to be defined in vertebrates. Here, we demonstrate that the conserved kinetochore-localized Ska1 complex tracks with depolymerizing microtubule ends and associates with both the microtubule lattice and curved protofilaments. In contrast, the Ndc80 complex, a central player in the kinetochore-microtubule interface, binds only to the straight microtubule lattice and lacks tracking activity. We demonstrate that the Ska1 complex imparts its tracking capability to the Ndc80 complex. Finally, we present a structure of the Ska1 microtubule-binding domain that reveals its interaction with microtubules and its regulation by Aurora B. This work defines an integrated kinetochore-microtubule interface formed by the Ska1 and Ndc80 complexes that associates with depolymerizing microtubules, potentially by interacting with curved microtubule protofilaments.

The Ska1 complex tracks with depolymerizing microtubule ends and induces the formation of curved microtubule structures

(A) Schematic of the experimental setup used to visualize the GFP-hSka1 complex binding to depolymerizing microtubules. Microtubules were induced to depolymerize by ablating a stabilizing GMPCPP cap with excitation light. (B) Representative kymographs showing the binding of GFP-hSka1 complex (left, 9 nM) and Ndc80 “Broccoli”-GFP (right, 9 nM) to depolymerizing microtubules (Dashed line indicates median protein-free microtubule depolymerization rate). Tracking events were identified as kymographs with substantial periods of an increased fluorescence signal at the microtubule end relative to the lattice. (C) Graph showing the normalized intensity of the GFP-hSka1 (n = 47-108) complex or GFP-Ndc80 complex (Mean ± SEM, n = 4–72) at the depolymerizing microtubule end over time. The Ska1 complex accumulates constantly at the depolymerizing end. (D) Depolymerization rates of microtubules in the presence and absence of a range of GFP-hSka1 complex and Ndc80 “Broccoli”-GFP concentrations (Mean ± SEM, n = 18–87). The presence of of 9 nM (P < 0.01) and 20 nM (P < 0.001) Ndc80 “Broccoli”-GFP significantly reduces the rate of microtubule depolymerization (Kruskal-Wallis test with Dunn’s post test), which is unaffected by similar concentrations of the Ska1 complex. Depolymerization rates were determined from the slopes of the corresponding kymographs. (E) TEM images of the dynamic microtubules in the absence and presence of 25 MgCl2, or the Ska1 and Ndc80 complexes (Scale bars, 100 nm). The Ska1, but not the Ndc80 complex, induces the formation of protofilament rings.

(A) The Ska1 complex and Ndc80 complex bind to microtubules synergistically. Quantification of microtubule co-sedimentation assays for hSka1 complex binding to taxol-stabilized microtubules alone and in the presence of a range of concentration of hNdc80 “Broccoli” (Mean ± SD, n = 3). (B) Quantification of microtubule pelleting assays of C. elegans Ndc80 complex binding to taxol-stabilized microtubules alone and in the presence of a range of ceSKA1 complex (n = 3). (C) Quantification of the fluorescence intensity (Mean ± SEM) on microtubules of hNdc80 “Broccoli”-GFP (blue) in the absence (n = 95) and presence of hSka1 (n = 136; P < 0.0001, t-test), and GFP-hSka1 (red) in the absence (n = 73) and presence of hNdc80 “Broccoli” (n = 81; P < 0.001, t-test). (D) Representative kymographs (microtubule position along horizontal axis, time along vertical axis) of time-lapse movies showing hNdc80 “Broccoli”-GFP (9 nM) binding to depolymerizing microtubules in the presence of unlabeled hSka1 complex (9 nM, n = 74). Tracking events were identified as kymographs with substantial periods of an increased fluorescence signal at the microtubule end relative to the remaining lattice. When both Ndc80 and Ska1 are present, the GMPCPP-caps are often converted into cargo and become transported by the shortening microtubules. (See also )